Grieu Fabienne, Joseph David, Norman Paul, Iacopetta Barry
Department of Radiation Oncology, Sir Charles Gairdner Hospital, University of Western Australia, Nedlands 6009, Australia.
Oncol Rep. 2004 Feb;11(2):501-4.
Single nucleotide polymorphisms (SNPs) in cancer-related genes can act as low risk genetic factors for the development of this disease. SNPs have also been shown to influence the efficacy and toxicity of various cytotoxic agents used in the treatment of cancer. Progress in these important areas of cancer research relies upon rapid, inexpensive and accurate means of SNP genotyping. In the present study we describe a fluorescent PCR-based method that utilizes single strand conformation polymorphism (SSCP) analysis to distinguish between different alleles. A real-time DNA fragment analysis platform and ultra-thin, re-useable gels allow short run times and hence a relatively high throughput to be achieved. A standardised procedure for the identification of optimal running conditions for each SNP is presented. We used this fluorescent-SSCP method to genotype SNPs in the MTHFR, p21, cyclin D1, MMP-2, vitamin D receptor, TNF-alpha and IL-6 genes. Multiplex PCR of two SNPs allows up to 500 genotypes per day to be evaluated with 100% accuracy. The low start-up and running costs make this method particularly well suited for SNP genotyping studies that involve up to 1,000 DNA samples.
癌症相关基因中的单核苷酸多态性（SNP）可作为该疾病发生的低风险遗传因素。SNP也已被证明会影响用于癌症治疗的各种细胞毒性药物的疗效和毒性。癌症研究这些重要领域的进展依赖于快速、廉价且准确的SNP基因分型方法。在本研究中，我们描述了一种基于荧光PCR的方法，该方法利用单链构象多态性（SSCP）分析来区分不同的等位基因。实时DNA片段分析平台和超薄、可重复使用的凝胶可实现较短的运行时间，从而实现相对较高的通量。本文还介绍了一种用于确定每个SNP最佳运行条件的标准化程序。我们使用这种荧光SSCP方法对MTHFR、p21、细胞周期蛋白D1、MMP-2、维生素D受体、TNF-α和IL-6基因中的SNP进行基因分型。对两个SNP进行多重PCR，每天可评估多达500个基因型，准确率达100%。较低的启动成本和运行成本使该方法特别适合涉及多达1000个DNA样本的SNP基因分型研究。
